The objective of this proposal is to understand the structural and mechanistic features governing the cyclization of farnesyl diphosphate (FPP) catalyzed by a homologous set of four plant sesquiterpene cyclases including Nictotiana tabacum 5-epi-aristolochene synthase (TEAS), Hyoscyamus muticus vetispiradiene synthase (HVS), Gossypium arboreum cadinene synthase (GCADS), and Artemisia annua L epi-cedrol synthase (AECS). During the previous funding period, the x-ray crystal structures of wide-type TEAS, mutant TEAS enzymes, and various small molecule complexes with each provided the first atomic resolution three- dimensional models of any plant terpene cyclase that allowed us to propose an enzymatic reaction mechanism consistent with the chemical rationalization of FPP cyclization to 5-epi-aristolochene. These three- dimensional guides also served as starting points for a mutagenesis strategy focused on a limited set of active site residues identified crystallographically. The initial set of site-directed mutants have given us solid evidence for the proposed reaction mechanism. We are now positioned to expand this directed approach combining structural and biochemical information with sequence alignments, homology modeling, and product identification to understand the structural and mechanistic basis for both substrate and product selectivity in terpene cyclases. The rapidly expanding database of plant terpene cyclase sequences gives us some indiction of what positions in the cyclase active site are most variable. We propose to use site-directed mutagenesis at single and multiple positions, steady state and pre-steady state kinetic analysis, product profiling by argentation thin-layer chromatography (arg-TLC) and radiometric gas chromatography (r-GC), product identification by gas chromatography/mass spectrometry (GC-MS), and x-ray crystallography to address the role that this variability plays in substrate selection ad in the alternative reaction mechanisms of related cyclases. This experimental strategy will serve as starting points for the rational manipulation of the substrate and product specificity in terpene cyclases. Modulation of the substrate and product specificity of these enzymes will directly impact efforts to produce novel compounds of both therapeutic and agricultural interest. Our current objective is to understand the regiochemical and stereochemical principles that govern the biosynthesis of pharmaceutically useful terpenoids.